University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2017-05-30 Investigating the Role of PIR1 and CD200R1 in the Innate Immune Response to Viral Pathogens Christopher R. MacKay University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Immunity Commons, Immunology of Infectious Disease Commons, Pathogenic Microbiology Commons, and the Virology Commons Repository Citation MacKay CR. (2017). Investigating the Role of PIR1 and CD200R1 in the Innate Immune Response to Viral Pathogens. GSBS Dissertations and Theses. https://doi.org/10.13028/M2602R. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/901 Creative Commons License This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. INVESTIGATING THE ROLE OF PIR1 AND CD200R1 IN THE INNATE IMMUNE RESPONSE TO VIRAL PATHOGENS A Dissertation Presented by CHRISTOPHER ROBERT MACKAY Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 30, 2017 M.D./Ph.D. Program INVESTIGATING THE ROLE OF PIR1 AND CD200R1 IN THE INNATE IMMUNE RESPONSE TO VIRAL PATHOGENS A Dissertation Presented by CHRISTOPHER ROBERT MACKAY The signatures of the Dissertation Defense Committee signifies completion and approval as to style and content of the Dissertation Evelyn A. Kurt-Jones, Ph.D., Thesis Advisor Robert Finberg M.D., Member of Committee Egil Lien, Ph.D., Member of Committee Craig Mello, Ph.D., Member of Committee Jennifer Wang, M.D., Member of Committee Kate Jeffrey, Ph.D. (Massachusetts General Hospital), Member of Committee The signature of the Chair of the Committee signifies that the written dissertation meets the requirements of the Dissertation Committee Neal Silverman, Ph.D., Chair of Committee The signature of the Dean of the Graduate School of Biomedical Sciences signifies that the student has met all graduation requirements of the school. Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences M.D./Ph.D. Program May 30, 2017 Abstract After initially being infected with a virus, before an adaptive immune response can be mounted, the innate immune system of a cell recognizes and responds to certain patterns present in pathogenic molecules. I studied the role of two genes—PIR1 and CD200R1—on the innate immune responses in two different mouse models of viral infection, infection with the picornavirus EMCV (encephalomyocarditis virus) and infection with HSV-1 (herpes simplex virus) in a mouse model of herpes simplex encephalitis, respectively. PIR1 is a putative RNA phosphatase that has been shown to play an important role in antiviral small RNA processing in C. elegans. It has also been shown to interact with the RIG-I-like receptor LGP2 in preliminary mammalian experiments. I sought to characterize the effect PIR1 has on the innate immune response to the virus EMCV in mice. By devel- oping a PIR1-null mouse, I have found that the role of PIR1 in the progression of EMCV in mice is limited. However, in vitro studies show that PIR1 might play an important role in regulating foreign RNA recognition during the earliest time points post-infection. CD200R1 is an anti-inflammatory signaling molecule that is expressed on myeloid- derived cells, and whose ligand is highly expressed within the central nervous system. I investigated the role of this receptor in an intracranial model of herpes simplex encephali- tis. CD200R1KO mice show improved survival following direct intracranial infection with HSV. I found this increased survival can be attributed to decreased levels of viral repli- cation in CD200R1KO compared to wild-type mice. Further investigation has shown that CD200R1 affects the signaling and upregulation of the pattern-recognition receptor TLR-2 (toll-like receptor 2), and thus CD200R1 may impact HSV-1 replication by affecting TLR2 signaling. Contents Abstract iii List of Tables viii List of Figures ix Abbreviations xi Chapter 1. Introduction 1 1.1 The Virus and the Human . 2 1.2 Host Responses to Viral Infection . 3 1.2.1 Pattern Recognition by the Innate Immune System . 3 Extracellular PAMP detection . 5 Toll-like receptor 2 . 5 Negative regulation of innate immune signaling . 6 CD200:CD200R1 signaling . 6 Intracellular response and Cytosolic RNA Receptors . 7 1.2.2 Antiviral small RNAs . 12 The antiviral small RNA response in C. elegans . 12 The antiviral small RNA response in D. melanogaster . 13 A mammalian antiviral small RNA response . 15 1.3 Conclusions . 18 Chapter 2. Investigating the role of PIR1 in the innate immune response to EMCV 19 2.1 Introduction . 20 2.1.1 PIR1 - protein that interacts with RBP 1 . 20 PIR1 in C. elegans ............................ 22 PIR1 in the mammalian system . 25 2.1.2 The replication cycle of Encephalomyocarditis Virus . 27 2.1.3 EMCV Pathogenesis in Mice . 29 EMCV Detection by the Innate Immune System . 30 2.1.4 Investigating the role of PIR1 in the mammalian innate immune re- sponse . 30 2.2 Materials and Methods . 33 2.2.1 Generating a PIR1 gene trap mouse strain . 33 2.2.2 Genotyping the PIR1 gene trap . 33 Genotyping via complementary DNA . 33 Genotyping via genomic DNA . 34 iv v Sequencing Intron 1 . 36 2.2.3 Mouse cell line generation . 36 Bone-marrow-derived dendritic cells . 36 2.2.4 Preparation of EMCV stocks . 41 2.2.5 EMCV plaque assays . 41 2.2.6 SNP analysis of mouse lineages . 42 2.2.7 Infection of mice with EMCV and collection of samples . 42 2.2.8 ELISA . 44 2.2.9 qRT-PCR . 45 2.2.10 Western blotting . 45 Preparing polyacrylamide gels . 46 Running a loaded gel . 46 Gel transfer . 46 Immunoblotting . 47 2.2.11 Confocal microscopy . 47 2.2.12 miRNA nanostring . 48 2.3 Results and Discussion . 49 2.3.1 Generating a PIR1-deficient mouse . 49 2.3.2 Characterizing the gene-trap allele . 51 2.3.3 EMCV infection in vivo ......................... 55 EMCV titers from heart and brain samples . 55 Inflammatory and viral RNA expression levels in heart and brain samples . 56 IFN-β expression in sera samples . 61 PIR1KO mice do not have a difference in survival rates following infection with EMCV. 61 2.3.4 Investigating the role of PIR1 in vitro . 61 PIR1-deficient cells show diminished RLR pathway activation . 64 2.3.5 The effect of PIR1 on small RNA pathways . 66 PIR1KO cells appear to have increased baseline expression of many miRNAs . 66 2.4 Conclusions . 70 2.4.1 Role of PIR1 in RLR signal transduction and/or ligand production . 70 The role of PIR1 with respect to its RNA phosphatase activity, and its antiviral role in C. elegans . 71 2.4.2 Combining the small RNA and IFN pathway phenotypes: a proposed model . 72 Chapter 3. CD200R1 supports HSV-1 viral replication and licenses pro- inflammatory signaling functions of TLR2. 74 3.1 Introduction . 75 3.1.1 Herpes Simplex Encephalitis . 75 The HSV Replication Lifecycle . 76 HSV and the Innate Immune System . 78 Mouse models of Herpes Simplex Encephalitis . 81 3.1.2 CD200:CD200R Signaling . 81 CD200:CD200R1 Signaling in CNS Infections . 82 CD200R1-/- mice are protected from HSV-1 encephalitis . 83 vi 3.2 Methods . 86 3.2.1 In vivo intracranial infection . 86 3.2.2 Immunohistochemistry staining for HSV . 86 3.2.3 Culturing Mouse Embryonic Fibroblast . 88 Isolation of Mouse Embryonic Fibroblasts . 88 Splitting MEFs . 88 Freezing MEFs . 89 Thawing MEFs . 89 3.2.4 Infection with GFP-expressing viruses, and measurement of infection by flow-cytometry . 89 3.2.5 Analysis of HSV protein expression by western blot . 90 3.2.6 Antibodies . 91 3.2.7 PCR Primers and Screening for CD200R1 Gene Targeting . 91 3.2.8 Exon-Specific screening. 92 3.2.9 Preparation and Stimulation of Peritoneal Macrophages . 92 3.2.10 Immunohistochemistry and Viral Particle Detection . 93 3.2.11 Histological Scoring of Brains . 94 3.2.12 Preparation of Viruses . 95 3.3 Results and Discussion . 97 3.3.1 CD200R1 -/- mice show similar levels of inflammation early in infection 97 3.3.2 CD200R1 is important in TLR2 mediated innate immune responses 100 3.4 Conclusions . 116 Chapter 4. Discussion 117 4.1 On the role of PIR1 in antiviral responses in mammals . 118 4.1.1 A potential role for PIR1 in the avRNAi anti-viral response . 119 Open questions on PIR1 and avRNAi in C. elegans . 119 Open questions on PIR1 and avRNAi in Drosphila . 120 A potential role of PIR1 in a mammalian avRNAi pathway . 121 A potential role of PIR1 in a mammalian miRNA pathway . 123 4.1.2 Future work and limitations in studying the avRNAi response in mam- mals . 124 The complex interaction between the type I IFN and avRNAi responses124 Separating the overlapping pathways of avRNAi and miRNA synthesis126 Single-cell pathway analysis . 127 4.1.3 Finding the relevance of avRNAi in mammals . 129 4.2 On the role of CD200R1 and TLR2 signaling in HSV replication . 131 4.2.1 NF-κB signaling and HSV replication . 131 4.2.2 TLR2 upregulation . 132 4.2.3 HSV signaling and TLR2 . 134 4.2.4 How does CD200R1 affect TLR2 signaling and upregulation? . 134 4.2.5 A general model for how CD200R1 affects TLR2 signaling and HSV-1 replication.